study-functions-activities-neuronal-k-cl-co-transporter-kcc2-using

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...

University of Exeter, Hatherly Laboratories

The mammalian SPS1-related proline/alanine-rich serine-threonine kinase SPAK (STK39) modulates ion transport across and between epithelial cells in response to environmental stimuli such osmotic stress and inflammation. Research over the last decade has established a central role for SPAK in the regulation of ion and water transport in the distal nephron, colonic crypts, and pancreatic ducts, and has implicated deregulated SPAK signaling in NaCl-sensitive hypertension, ulcerative colitis and Crohn's disease, and cystic fibrosis. Areas covered: We review recent advances in our understanding of the role of SPAK kinase in the regulation of epithelial transport. We highlight how SPAK signaling - including its upstream Cl sensitive activators, the WNK kinases, and its downstream ion transport targets, the cation- Cl cotransporters contribute to human disease. We discuss prospects for the pharmacotherapeutic targeting of SPAK kinase in specific human disorders that feature impaired epithelial homeostasis. Expert opinion: The development of novel drugs that antagonize the SPAK-WNK interaction, inhibit SPAK kinase activity, or disrupt SPAK kinase activation by interfering with its binding to MO25α/β could be useful adjuncts in essential hypertension, inflammatory colitis, and cystic fibrosis.

Pharmacological targeting of SPAK kinase in disorders of impaired epithelial transport.

The choroid plexus epithelium (CPE) secretes higher volumes of fluid (cerebrospinal fluid, CSF) than any other epithelium and simultaneously functions as the blood-CSF barrier to gate immune cell entry into the central nervous system. Posthemorrhagic hydrocephalus (PHH), an expansion of the cerebral ventricles due to CSF accumulation following intraventricular hemorrhage (IVH), is a common disease usually treated by suboptimal CSF shunting techniques. PHH is classically attributed to primary impairments in CSF reabsorption, but little experimental evidence supports this concept. In contrast, the potential contribution of CSF secretion to PHH has received little attention. In a rat model of PHH, we demonstrate that IVH causes a Toll-like receptor 4 (TLR4)- and NF-κB-dependent inflammatory response in the CPE that is associated with a ∼3-fold increase in bumetanide-sensitive CSF secretion. IVH-induced hypersecretion of CSF is mediated by TLR4-dependent activation of the Ste20-type stress kinase SPAK, which binds, phosphorylates, and stimulates the NKCC1 co-transporter at the CPE apical membrane. Genetic depletion of TLR4 or SPAK normalizes hyperactive CSF secretion rates and reduces PHH symptoms, as does treatment with drugs that antagonize TLR4-NF-κB signaling or the SPAK-NKCC1 co-transporter complex. These data uncover a previously unrecognized contribution of CSF hypersecretion to the pathogenesis of PHH, demonstrate a new role for TLRs in regulation of the internal brain milieu, and identify a kinase-regulated mechanism of CSF secretion that could be targeted by repurposed US Food and Drug Administration (FDA)-approved drugs to treat hydrocephalus.

Inflammation-dependent cerebrospinal fluid hypersecretion by the choroid plexus epithelium in posthemorrhagic hydrocephalus.

The K-Cl co-transporter KCC2 (SLC12A5) tunes the efficacy of GABA receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl]. KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABA receptor (GABAR)-mediated transmission in mature hippocampal neurons. Enhancing GABAR-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl-sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl] to GABAR activity.

GABA receptor dependent synaptic inhibition rapidly tunes KCC2 activity via the Cl-sensitive WNK1 kinase.

Omega-3 fatty acids are products of secondary metabolism, essential for growth and important for human health. Although there are numerous reports of bacterial production of omega-3 fatty acids, less information is available on the biotechnological production of these compounds from bacteria. The production of eicosapentaenoic acid (EPA, 20:5ω3) by a new species of marine bacteria Shewanella electrodiphila MAR441T was investigated under different fermentation conditions. This strain produced a high percentage (up to 26%) of total fatty acids and high yields (mg / g of biomass) of EPA at or below the optimal growth temperature. At higher growth temperatures these values decreased greatly. The amount of EPA produced was affected by the carbon source, which also influenced fatty acid composition. This strain required Na+ for growth and EPA synthesis and cells harvested at late exponential or early stationary phase had a higher EPA content. Both the highest amounts (20 mg g-1) and highest percent EPA content (18%) occurred with growth on L-proline and (NH4)2SO4. The addition of cerulenin further enhanced EPA production to 30 mg g-1. Chemical mutagenesis using NTG allowed the isolation of mutants with improved levels of EPA content (from 9.7 to 15.8 mg g-1) when grown at 15°C. Thus, the yields of EPA could be substantially enhanced without the need for recombinant DNA technology, often a commercial requirement for food supplement manufacture.

Enhanced eicosapentaenoic acid production by a new deep-sea marine bacterium Shewanella electrodiphila MAR441T.

Brain computations rely on a proper balance between excitation and inhibition which progressively emerges during postnatal development in rodent. γ-Aminobutyric acid (GABA) neurotransmission supports inhibition in the adult brain but excites immature rodent neurons. Alterations in the timing of the GABA switch contribute to neurological disorders, so unveiling the involved regulators may be a promising strategy for treatment. Here we show that the adipocyte hormone leptin sets the tempo for the emergence of GABAergic inhibition in the newborn rodent hippocampus. In the absence of leptin signaling, hippocampal neurons show an advanced emergence of GABAergic inhibition. Conversely, maternal obesity associated with hyperleptinemia delays the excitatory to inhibitory switch of GABA action in offspring. This study uncovers a developmental function of leptin that may be linked to the pathogenesis of neurological disorders and helps understanding how maternal environment can adversely impact offspring brain development.

The adipocyte hormone leptin sets the emergence of hippocampal inhibition in mice.

Normal vascular development includes the formation and specification of arteries, veins, and intervening capillaries. Vein of Galen malformations (VOGMs) are among the most common and severe neonatal brain arterio-venous malformations, shunting arterial blood into the brain's deep venous system through aberrant direct connections. Exome sequencing of 55 VOGM probands, including 52 parent-offspring trios, revealed enrichment of rare damaging de novo mutations in chromatin modifier genes that play essential roles in brain and vascular development. Other VOGM probands harbored rare inherited damaging mutations in Ephrin signaling genes, including a genome-wide significant mutation burden in EPHB4. Inherited mutations showed incomplete penetrance and variable expressivity, with mutation carriers often exhibiting cutaneous vascular abnormalities, suggesting a two-hit mechanism. The identified mutations collectively account for ∼30% of studied VOGM cases. These findings provide insight into disease biology and may have clinical implications for risk assessment.

Mutations in Chromatin Modifier and Ephrin Signaling Genes in Vein of Galen Malformation.

In recent years, cation-chloride cotransporters (CCCs) have drawn attention in the medical neuroscience research. CCCs include the family of Na-coupled Cl importers (NCC, NKCC1, and NKCC2), K-coupled Cl exporters (KCCs), and possibly polyamine transporters (CCC9) and CCC interacting protein (CIP1). For decades, CCCs have been the targets of several commonly used diuretic drugs, including hydrochlorothiazide, furosemide, and bumetanide. Genetic mutations of NCC and NKCC2 cause congenital renal tubular disorders and lead to renal salt-losing hypotension, secondary hyperreninemia, and hypokalemic metabolic alkalosis. New studies reveal that CCCs along with their regulatory WNK (Kinase with no lysine (K)), and SPAK (Ste20-related proline-alanine-rich kinase)/OSR1(oxidative stress-responsive kinase-1) are essential for regulating cell volume and maintaining ionic homeostasis in the nervous system, especially roles of the WNK-SPAK-NKCC1 signaling pathway in ischemic brain injury and hypersecretion of cerebrospinal fluid in post-hemorrhagic hydrocephalus. In addition, disruption of Cl exporter KCC2 has an effect on synaptic inhibition, which may be involved in developing pain, epilepsy, and possibly some neuropsychiatric disorders. Interference with KCC3 leads to peripheral nervous system neuropathy as well as axon and nerve fiber swelling and psychosis. The WNK-SPAK/OSR1-CCCs complex emerges as therapeutic targets for multiple neurological diseases. This review will highlight these new findings.

The WNK-SPAK/OSR1 Kinases and the Cation-Chloride Cotransporters as Therapeutic Targets for Neurological Diseases.

KCC2 is a vital neuronal K/Cl cotransporter that is implicated in the etiology of numerous neurological diseases. In normal cells, KCC2 undergoes developmental dephosphorylation at Thr and Thr We engineered mice with heterozygous phosphomimetic mutations T906E and T1007E ( ) to prevent the normal developmental dephosphorylation of these sites. Immature (postnatal day 15) but not juvenile (postnatal day 30)  mice exhibited altered GABAergic inhibition, an increased glutamate/GABA synaptic ratio, and greater susceptibility to seizure.  mice also had abnormal ultrasonic vocalizations at postnatal days 10 to 12 and impaired social behavior at postnatal day 60. Postnatal bumetanide treatment restored network activity by postnatal day 15 but failed to restore social behavior by postnatal day 60. Our data indicate that posttranslational KCC2 regulation controls the GABAergic developmental sequence in vivo, indicating that deregulation of KCC2 could be a risk factor for the emergence of neurological pathology.

Impaired regulation of KCC2 phosphorylation leads to neuronal network dysfunction and neurodevelopmental pathology.

The SLC12A cation-Cl cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.

Modulation of brain cation-Cl cotransport via the SPAK kinase inhibitor ZT-1a.

Hypertension is the most prevalent health condition worldwide, affecting ~1 billion people. Gordon's syndrome is a form of secondary hypertension that can arise due to a number of possible mutations in key genes that encode proteins in a pathway containing the With No Lysine [K] (WNK) and its downstream target kinases, SPS/Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1). This pathway regulates the activity of the thiazide-sensitive sodium chloride cotransporter (NCC), which is responsible for NaCl reabsorption in the distal nephron. Therefore, mutations in genes encoding proteins that regulate the NCC proteins disrupt ion homeostasis and cause hypertension by increasing NaCl reabsorption. Thiazide diuretics are currently the main treatment option for Gordon's syndrome. However, they have a number of side effects, and chronic usage can lead to compensatory adaptations in the nephron that counteract their action. Therefore, recent research has focused on developing novel inhibitory molecules that inhibit components of the WNK-SPAK/OSR1-NCC pathway, thereby reducing NaCl reabsorption and restoring normal blood pressure. In this review we provide an overview of the currently reported molecular inhibitors of the WNK-SPAK/OSR1-NCC pathway and discuss their potential as treatment options for Gordon's syndrome.

WNK-SPAK/OSR1-NCC kinase signaling pathway as a novel target for the treatment of salt-sensitive hypertension.

Editorial: Marine Microbial-Derived Molecules and Their Potential Medical and Cosmetic Applications.

The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline-alanine-rich kinase (SPAK) are physiological substrates of the with-no-lysine (WNK) kinase. They are the master regulators of cation Cl cotransporters that could be targeted for discovering novel antihypertensive agents. Both kinases have a conserved carboxy-terminal (CCT) domain that recognizes a unique peptide motif (Arg-Phe-Xaa-Val) present in their upstream kinases and downstream substrates. Here, we have combined molecular docking with molecular dynamics simulations and free-energy calculations to identify potential inhibitors that can bind to the allosteric pocket of the OSR1-CCT domain and impede its interaction with the WNK peptide. Our study revealed that STOCK1S-14279 and Closantel bound strongly to the allosteric pocket of OSR1 and displaced the WNK peptide from the primary pocket of OSR1. We showed that primarily Arg1004 and Gln1006 of the WNK4-peptide motif were involved in strong H-bond interactions with Glu453 and Arg451 of OSR1. Besides, our study revealed that atoms of Arg1004 were solvent-exposed in cases of STOCK1S-14279 and Closantel, implying that the WNK4 peptide was moved out of the pocket. Overall, the predicted potential inhibitors altogether abolish the OSR1-WNK4-peptide interaction, suggesting their potency as a prospective allosteric inhibitor against OSR1.

Molecular Mechanism of Inhibiting WNK Binding to OSR1 by Targeting the Allosteric Pocket of the OSR1-CCT Domain with Potential Antihypertensive Inhibitors: An  Study.

Article	Year
Pharmacological targeting of SPAK kinase in disorders of impaired epithelial transport. Expert opinion on therapeutic targets\| PubMed ID: 28679296
Inflammation-dependent cerebrospinal fluid hypersecretion by the choroid plexus epithelium in posthemorrhagic hydrocephalus. Nature medicine\| PubMed ID: 28692063
GABA receptor dependent synaptic inhibition rapidly tunes KCC2 activity via the Cl-sensitive WNK1 kinase. Nature communications\| PubMed ID: 29176664
Enhanced eicosapentaenoic acid production by a new deep-sea marine bacterium Shewanella electrodiphila MAR441T. PloS one\| PubMed ID: 29176835
The adipocyte hormone leptin sets the emergence of hippocampal inhibition in mice. eLife\| PubMed ID: 30106375
Mutations in Chromatin Modifier and Ephrin Signaling Genes in Vein of Galen Malformation. Neuron\| PubMed ID: 30578106
The WNK-SPAK/OSR1 Kinases and the Cation-Chloride Cotransporters as Therapeutic Targets for Neurological Diseases. Aging and disease\| PubMed ID: 31165006
Impaired regulation of KCC2 phosphorylation leads to neuronal network dysfunction and neurodevelopmental pathology. Science signaling\| PubMed ID: 31615899
Modulation of brain cation-Cl cotransport via the SPAK kinase inhibitor ZT-1a. Nature communications\| PubMed ID: 31911626	2020
WNK-SPAK/OSR1-NCC kinase signaling pathway as a novel target for the treatment of salt-sensitive hypertension. Acta pharmacologica Sinica\| PubMed ID: 32724175	2021
Editorial: Marine Microbial-Derived Molecules and Their Potential Medical and Cosmetic Applications. Frontiers in microbiology\| PubMed ID: 34335540
Molecular Mechanism of Inhibiting WNK Binding to OSR1 by Targeting the Allosteric Pocket of the OSR1-CCT Domain with Potential Antihypertensive Inhibitors: An Study. The journal of physical chemistry. B\| PubMed ID: 34369793

Jinwei Zhang

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